Pharmaceutical composition which improves in vivo gene transfer

ABSTRACT

A pharmaceutical composition, including at least one nucleic acid, a Tyrode&#39;s medium having 140 mM NaC1, 6 mM KC1, 3 mM CaC1 2 , 2 mM MgC1 2 , 10 mM Hepes, pH 7.4, and 10 mM glucose; and a tetrafunctional copolymer of formula (I): 
                         
where x and y represent, independently of one another, an integer of between 1 and 500, the tetrafunctional copolymer is in a form of a mineral salt wherein said tetrafunctional copolymer is in a cationic form. The compound of formula (I) has a molecular weight of between 1,000 and 25,000 g/mol and an EO/PO ratio of 0.8 to 1.2.

This is a Continuation of application Ser. No. 10/502,973 filed Mar. 8,2005, now issued as U.S. Pat. No. 7,709,452, which is a National StageApplication of PCT/FR03/00407 filed Feb. 7, 2003. The entire disclosureof the prior applications are hereby incorporated by reference in theirentirety.

The present invention is directed, in a main capacity, toward apharmaceutical composition for facilitating the cellular transfer ofnucleic acid(s) and more particularly into muscle or cardiac cells invivo.

The problem under consideration in the context of the present inventionis that of the transfer of gene information into a cell, and moreparticularly into its nucleus, in order for the gene under considerationto be translated therein into protein.

Currently, two technologies are mainly used for cellular gene transfer.The first uses adeno-associated virus, AAV, which is a recombinantvirus. As regards the second, it is electrotransfer, which consists inimposing an electric field on the cells under consideration, afterhaving injected the DNA into them. Although these two methods make itpossible to obtain satisfactory levels of gene transfer efficiency, theyhave, on the other hand, a disadvantage in terms of toxicity. What ismore, AAV today raises many technical difficulties in terms of itspreparation, purification and/or characterization, and the potentialrisk of integration into the host cell's genome could bring aboutcancerization phenomena. Finally, gene transfer into the cardiac musclecannot be obtained by means of the electrotransfer technique forsurvival reasons.

In this case, the present invention is precisely advantageous fortransfection into skeletal, smooth and cardiac muscle cells.

As regards synthetic vectors, like cationic lipids for example, theyprove to be inefficient in vivo. Only naked DNA is capable of resultingin the expression of a protein after injection into skeletal and cardiacmuscle. Unfortunately, the amount of protein synthesized afterintramuscular injection of naked DNA remains insufficient to envisionclinical applications. In fact, one of the major problems of using nakedDNA for gene transfer into muscle tissues is its poor efficiency, whichis not improved by increasing amounts of injected DNA. Now, for reasonsof convenience, in particular in terms of accessibility, it would beparticularly advantageous to favor the expression of proteins of localor systemic therapeutic interest in these muscles.

Consequently, there exists today a real need for a vector that is idealin gene therapy, i.e. a nontoxic vector which does not induce an immunereaction and which makes it possible to obtain optimal expression ofprotein, the therapeutic effect of which is desired.

The object of the present invention is precisely to propose the use ofspecific chemical molecules for significantly increasing the efficiencyof transfection of DNA with which they are combined.

More precisely, the present invention relates to a pharmaceuticalcomposition, characterized in that it combines with at least one nucleicacid a tetrafunctional copolymer of formula (I):

or one of its organic or mineral salts or derivatives, in which x and yrepresent, independently of one another, an integer of between 1 and 500with x having a value such that said molecule comprises at least 30% byweight of ethylene oxide units, and in which the compound of formula (I)is preferably used in cationic form.

The abovementioned molecules, which are specific poloxamines, consist ofhydrophobic segments (propylene oxide bearing the y indices), ofhydrophilic segments (ethylene oxide bearing the x indices) and of apositively charged central ethylenediamine component (NCH₂—CH₂N).

The inventors have more particularly demonstrated that thesepoloxamines, which were until then presented as nonionic polymers (WO00/47186), can in fact take a cationic form.

Surprisingly, the appearance of these positive charges on the centralethylenediamine component does not in any way harm the efficiency ofcellular transfer, on the contrary. In fact, cationic chemical moleculesof the cationic lipid, protein and cationic peptide type do not improve,with respect to naked DNA, gene transfer in muscle tissues (Lluis M. M.et al., 1999, Proc. Natl. Acad. Sci., vol. 96, pp. 4262-4267).

A particular subject of the invention is therefore a pharmaceuticalcomposition, characterized in that it combines with at least one nucleicacid at least one organic or mineral salt of the tetrafunctionalcopolymer of formula (I):

used in a cationic form,

in which x and y represent, independently of one another, an integer ofbetween 1 and 500 with x having a value such that said moleculecomprises at least 30% by weight of ethylene oxide units.

The compound of general formula (I) is therefore preferably used in theform of one of its salts, and more preferably in a cationic form. To dothis, the composition claimed combines with said compound a preferablymineral salt, and more preferably an alkali metal salt or analkaline-earth metal salt. It may in particular be chosen from sodiumchloride, potassium chloride or lithium chloride and sodium thiocyanate,or more preferably calcium chloride (CaCl₂) or magnesium chloride(MgCl₂).

This salt may be introduced in isotonic, hypotonic or hypertonic amount.

The inventors have also established the advantage of controlling the pHand/or the ionic composition of the formulation, in order to be surethat the copolymer of formula (I) is in its cationic form.

A pH of 6.5 to 8, preferably 7 to 7.8, more preferably 7.4, proves to beparticularly advantageous.

According to a preferred embodiment of the invention, the composition isformulated in a medium referred to as Tyrode's (medium containing 3 mMCaCl₂, 2 mM MgCl₂, 6 mM KCl, 140 mM NaCl, 10 mM glucose and 10 mM Hepes,pH 7.4) (Tyrode Pharmacology. Philadelphia, 1908, 2nd edition, 1912).The presence of the Tyrode's makes it possible in particular to controlthe ionic composition of the formulation and the pH and, consequently,the use of the compound of formula (I) in a cationic form.

Without wishing to be bound to any mechanism of action, the inventors infact observe that the poloxamines used in a cationic form, in particularin the presence of Tyrode medium, make it possible to condense the DNA.

Unexpectedly, the inventors have also demonstrated that the compounds ofgeneral formula (I), in which the ethylene oxide units are present in aproportion of at least 30% by weight, prove to be particularly efficientfor the transfer of an associated gene in vivo. This efficiency is inparticular illustrated by example 1 hereinafter. According to apreferred variant, the compounds according to the invention comprise nomore than 85% by weight of ethylene oxide units. More preferably, theyhave approximately between 35 and 50%, and preferably approximately 40%,by weight of ethylene oxide units.

According to a preferred variant of the invention, the molecules ofcompounds of general formula (I) also have a molecular weight of atleast 800 g/mol, and more preferably of between 1000 and 25 000 g/mol.

According to a preferred embodiment of the invention, the compounds ofgeneral formula have an EO/PO unit ratio of between 0.5 and 1.5, andpreferably of the order of 1±0.2.

As compounds of general formula (I) that are most particularly suitablefor the present invention, mention may more particularly be made ofmolecules having, respectively, a molecular weight of 1650 g for anEO/PO ratio of 15:16 (for example poloxamine 304), of 5500 g/mol for anEO/PO ratio of 50:56 (for example poloxamine 704) and of 6700 g/mol foran EO/PO ratio of 61:68 (for example poloxamine 904).

For the purpose of the present invention, the term “derivative” isintended to cover compounds which have the chemical structure of generalformula I but which also carry secondary chemical or biologicalfunctions or entities capable of conferring on them complementaryproperties. Particularly representative of these derivatives arecompounds of general formula I in which there is also as least oneintra- or extracellular targeting unit. By way of nonlimitingillustration of these targeting units, mention may more particularly bemade of peptides carrying a nuclear localization sequence, or peptideswhich recognize receptors present at the surface of certain cells.

According to a preferred embodiment, the composition is free of sodiumphosphate and/or of glucose.

For the purpose of the present invention, the term “nucleic acid” coversboth a deoxyribonucleic acid and a ribonucleic acid.

In this case, they may be sequences of natural or artificial origin, andin particular genomic DNA, cDNA, mRNA, tRNA, rRNA, small interferenceRNA (iRNA) hybrid sequences, or synthetic or semi-synthetic sequences ofoligonucleotides which may or may not have been modified. These nucleicacids may be of human, animal, plant, bacterial, viral, etc. origin.They may be obtained by any technique known to those skilled in the art,and in particular by screening libraries, by chemical synthesis or bymixed methods including the chemical or enzymatic modification ofsequences obtained by screening libraries. They may be chemicallymodified.

As regards more particularly the deoxyribonucleic acids, they may besingle-stranded or double-stranded, just as short oligonucleotides orlonger sequences. These deoxyribonucleic acids can carry therapeuticgenes, regulatory sequences for transcription or for replication,modified or unmodified antisense sequences, regions for binding to othercellular components, etc. They may in particular direct the synthesis ofa polypeptide specific for an infectious agent or may be capable ofremedying a genetic or acquired deficiency.

For the purpose of the invention, the term “therapeutic gene” isintended to mean in particular any gene encoding a protein producthaving a therapeutic effect. The protein product thus encoded may be aprotein, a peptide, etc. This protein product may be homologous withrespect to the target cell (i.e. a product which is normally expressedin the target cell when said cell exhibits no pathology). In this case,the expression of a protein makes it possible, for example, tocompensate for an insufficient expression in the cell or the expressionof a protein that is inactive or weakly active due to a modification, orelse to overexpress said protein. The therapeutic gene can also encode amutant of a cellular protein, having increased stability, modifiedactivity, etc. The protein product may also be heterologous with respectto the target cell. In this case, an expressed protein may, for example,add to or introduce a deficient activity in or into the cell, allowingit to combat a pathology or simulate an immune response.

Among the therapeutic products for the purpose of the present invention,mention may be made more particularly of enzymes, blood derivatives,hormones, lymphokines such as interleukins, interferons, TNF, etc.,growth factors such as vascular endothelial growth factor, insulin-likegrowth factor and fibroblast growth factor, neurotransmitters or theirprecursors or enzymes for synthesizing them, trophic factors such asBDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5 and HARP/pleiotrophin,dystrophin or a mini-dystrophin, utrophin, the cystic fibrosis-relatedCFTR protein, tumor suppressor genes such as p53, Rb, Rap1A, DCC andk-rev, genes encoding factors involved in clotting, such as factors VII,VIII and IX, genes involved in DNA repair, suicide genes (thymidinekinase, cytosine deaminase), genes for hemoglobin or other proteintransporters, genes corresponding to the proteins involved in lipidmetabolism, of apolipo-protein type chosen from apolipoproteins A-I,A-II, A-IV, B, C-I, C-II, C-III, D, E, F, G, H, J and Apo(A), metabolicenzymes such as, for example, lipoprotein lipase, hepatic lipase,lecithin cholesterol acyl-transferase, cholesterol 7-alpha-hydroxylase,phosphatidic acid phosphatase or lipid-transfer proteins such ascholesteryl ester transfer protein and phospholipid transfer protein, anHDL-binding protein or else a receptor chosen, for example, from LDLreceptors, chylomicron-remnant receptors and scavenger receptors,erythropoietin, protein kinase C-3, etc.

The therapeutic nucleic acid may also be an antisense gene or sequence,the expression of which in the target cell makes it possible to controlgene expression or cellular mRNA transcription. Such sequences may, forexample, be transcribed, in the target cell, into RNAs that arecomplementary to cellular mRNAs, and thus block the translation thereofinto protein. The therapeutic genes also comprise the sequences encodingribozymes, which are capable of selectively destroying target RNAs.

As indicated above, the nucleic acid may also comprise one or more genesencoding an antigenic peptide capable of generating an immune responsein humans or animals. In this particular embodiment, the inventiontherefore makes it possible to produce either vaccines orimmuno-therapeutic treatments applied to humans or to animals, inparticular against microorganisms, viruses or cancers. They may inparticular be antigenic peptides specific for the Epstein Barr virus,for the HIV virus, for the hepatitis B virus, for the pseudorabiesvirus, for the syncitia forming virus or for other viruses, or elsespecific for tumors.

Preferably, the nucleic acid also comprises sequences which allow theexpression of the therapeutic gene and/or of the gene encoding theantigenic peptide in the desired cell or organ. They may be thesequences which are naturally responsible for the expression of the geneunder consideration when these sequences are capable of functioning inthe infected cell. They may also be sequences of different origin(responsible for the expression of other proteins, or even syntheticsequences). In particular, they may be promoter sequences for eukaryoticor viral genes. For example, they may be promoter sequences derived fromthe genome of the cell that it is desired to infect. Similarly, they maybe promoter sequences derived from the genome of a virus. In thisregard, mention may be made, for example, of the promoters of the E1A,MLP, CMV and RSV genes, and tissue-specific promoters such as myosinchain promoters, for example, etc. In addition, these expressionsequences can be modified by the addition of activation sequences,regulatory sequences, etc. They may also involve an inducible orrepressible promoter.

Moreover, the nucleic acid can also comprise, in particular upstream ofthe therapeutic gene, a signal sequence which directs the synthesizedtherapeutic product into the target cell's secretion pathways. Thissignal sequence may be the natural signal sequence of the therapeuticproduct, but it may also be any other functional signal sequence, or anartificial signal sequence. The nucleic acid can also comprise a signalsequence which directs the synthesized therapeutic product to aparticular compartment of the cell.

Besides the compound of general formula (I), the claimed compositionscan comprise one or more adjuvant(s), and in particular a surfactant.

By way of representation of these adjuvants, mention may moreparticularly be made of celluloses, such as carboxymethylcellulose orhydroxypropylcellulose, hyaluronate or alginate salts, pectins,polyethylene glycols, dextrans, polyvinylpyrrolidones, chitosans,polyvinyl alcohols, propylene glycols, polyvinyl acetates, lecithins,polylactic and polyhydroxybutyric acids, and poloxamers of thePluronics® (PEO-PPO-PEO) and reverse Pluronics® (PPO-PEO-PPO) series.

The compositions according to the invention may also use one or moretargeting elements for directing the nucleic acid complexes to receptorsor ligands at the surface of the cell. By way of example, thecomposition of the present invention may comprise one or more antibodiesdirected against cell surface molecules, or else one or moremembrane-receptor ligands such as insulin, transferrin, folic acid orany other growth factor, cytokines or vitamins. Advantageously, thecomposition can use modified or unmodified lectins in order to targetspecific polysaccharides at the surface of the cell or on theneighboring extracellular matrix. Proteins containing an RGD unit,peptides containing a tandem of RGD units, which may or may not becyclic, and also polylysine peptides or ligand peptides, which may benatural or synthetic, can thus be used.

The compound of general formula (I) may be incorporated in a proportionof 0.005% to 15% by weight/volume of said composition, and morepreferably between 0.01% and 10% by weight/volume.

The claimed compositions are obtained by mixing the nucleic acid underconsideration with at least one compound of general formula (I) in asolution compatible with in vivo administration. By way of illustration,these compositions may be prepared using the following protocol: asolution containing the nucleic acid (in particular DNA) two-timesconcentrated in 300 mM NaCl or 2× (two-times concentrated) Tyrode's anda two-times more concentrated aqueous solution containing a compound ofgeneral formula (I) are mixed volume for volume. The entire mixture isstirred, preferably by means of a VORTEX.

It has thus been noted that the claimed compounds of general formula (I)make it possible to increase the amount of protein synthesized by themuscle by a factor ranging from 5 to 20, compared to naked DNA.Moreover, with the composition according to the invention, no expressionof a transgene has been noted in cells in culture in vitro, usingconventional cell culturing methods.

The pharmaceutical compositions of the invention preferably contain avehicle that is pharmaceutically acceptable for an injectableformulation, in particular for injection directly into the desired organor for topical administration, for example to the skin and/or mucousmembranes. They may in particular be sterile isotonic solutions or dry,in particular lyophilized, compositions which, by means of the addition,according to the case, of sterilized water or of physiological saline,make it possible to constitute injectable solutes.

It is clear that the doses of nucleic acid used for the injection andalso the number of administrations can be adjusted by means of variousparameters, and in particular as a function of the method ofadministration under consideration, of the pathology involved, of thenature of the gene to be expressed or of the desired duration oftreatment.

As regards more particularly the method of administration, it mayinvolve either direct injection into the tissues or the circulatorysystem, or treatment of cells in culture followed by reimplantation invivo by injection or graft.

The claimed composition proves to be particularly advantageous forinternal administration.

For the purpose of the present invention, the term “internaladministration” signifies that the claimed compositions are compatiblewith administration into the tissue of an organism, for example amuscle, intra-dermally or subcutaneously. Furthermore, topical, oral,pulmonary, nasal and mucosal, such as, for example, buccal, vaginal orrectal, administration may be used.

The compositions according to the invention are particularlyadvantageous from a therapeutic point of view.

Thus, the potential applications are in the field of gene therapy and inparticular in the production by a muscle tissue of a protein of local orsystemic therapeutic interest.

In fact, after gene transfer into the muscle, in the presence of atleast one molecule of general formula (I), this organ can serve as areservoir for the synthesis of heterologous proteins which will acteither locally (angiogenic factor, etc.) or systemically (clottingfactor, growth factor, insulin, etc.).

What is more, the claimed composition may make it possible to abolishthe plateau effect obtained with naked DNA. Specifically, when theamount of DNA injected into the muscle increases, the transfectionincreases in a linear fashion up to a certain dose, and then the amountof protein expressed reaches a maximum. On the other hand, withformulations in accordance with the invention, the amount of proteinexpressed increases exponentially with the increase in the amount of DNAinjected into the muscle.

Another application comes from the field of immunization. In this case,a DNA encoding a bacterial, viral or other antigen is injected intocells, preferably muscle cells. Insofar as the claimed compositions areparticularly advantageous for increasing the amount of proteinssynthesized by the transfected cells, it is possible, by virtue of this,to obtain higher concentrations of antibodies and of cytotoxic Tlymphocytes.

A subject of the present invention is also the use of a compound ofgeneral formula I or of one of its organic or mineral salts, as a vectorfor the cellular transfer in vivo of at least one nucleic acid asdefined according to the invention.

The use of a compound of general formula I as defined above, forpreparing a composition intended to provide the cellular transfer invivo of at least one nucleic acid, is also within the scope of theinvention.

The invention also relates to a method for the cellular transfection invivo of at least one nucleic acid, characterized in that said nucleicacid is administered together with at least one compound of generalformula I as defined above or one of its organic or mineral salts.

The administration can be carried out topically, directly into the cellsunder consideration, or by means of one of the routes of administrationdiscussed above.

According to a preferred variant of the invention, the target cells aremuscle cells or cardiac cells.

According to a preferred embodiment of the invention, poloxamine 304 isused as a vector for transferring a nucleic acid, in vivo, into muscle,or especially cardiac, cells.

Advantageously, the poloxamine 304 is formulated in a compositioncontaining Tyrode's medium.

The present invention will be described more fully by means of theexamples and figures which follow, which should be considered asnonlimiting illustrations.

FIGURES

FIG. 1: Expression of luciferase after intramuscular injection of DNAformulated according to the invention.

FIG. 2: Macroscopic visualization of β-galactosidase activity afterinjection, into the mouse anterior tibialis, of DNA formulated accordingto the invention.

FIG. 3: Visualization by fluorescence microscopy of GFP expression in amuscle cell.

FIG. 4: Representation of β-galactosidase expression in the mouseanterior tibialis after injection, by electrotransfer, of said DNA or ofsaid DNA formulated according to the invention.

FIG. 5: Visualization by cryoelectron microscopy. The 10% poloxamine 904is mixed with DNA at 0.15 mg/ml, either in 150 mM NaCl (A, B) or inTyrode's (140 mM NaCl, 6 mM KCl, 3 mM CaCl₂, 2 mM MgCl₂, 10 mM Hepes, pH7.4, and 10 mM glucose) (C, D, E). Scale bar 100 nm.

MATERIALS

The compounds of general formula (I) tested are listed below in table 1,which records their physicochemical characteristics.

TABLE 1 Number of Number of Poloxamine MW % ethylene ethylene propylenemolecule (g/mol) oxide oxides oxides EO/PO 304 1650 40 15 16 0.93 7045500 40 50 56 0.89 904 6700 40 61 68 0.89 908 25 000   80 454 85 5.3

EXAMPLE 1 In vivo Transfection of a Gene into the Muscle in the Presenceof a Compound of General Formula (I)

Sample Preparation:

Two concentrations optimized for each of the products tested wereconsidered.

Each trial uses 15 μg of DNA in 50 μl of 150 mM NaCl combined with twodifferent concentrations for a compound of general formula (I) that istested.

The concentrations tested are, for compound 904, respectively 0.01% and0.1%, for compound 704: 0.25% and 0.5%, and for compound 304: 5% and10%.

A control sample of DNA is formulated in the absence of compounds ofgeneral formula (I).

Each sample thus formulated is injected to the anterior tibialis ofseven-week-old Swiss mice. After seven days, the mice are sacrificed andthe injected anterior tibialis is dissected and then ground in a lysisbuffer in the presence of a cocktail of protease inhibitors from RocheDiagnostics. The luciferase activity is assayed in the supernatant byluminometry (vector 2 from Perkin Elmer, Les Ulis, France) using theLuciferase Assay System® assay kit distributed by Promega(Charbonnières, France).

FIG. 1 reports the results obtained.

It is noted that the presence of a compound of general formula (I) makesit possible to significantly improve the expression of the luciferaseactivity by a factor ranging from 10 to 15.

EXAMPLE 2 Transfer of a Gene Encoding β-Galactosidase into a SkeletalMuscle

In the same way as in example 1, an injection of naked DNA (A), or offormulations in accordance with the invention containing 5% (B) or 10%of compound 304 (C), or 0.01% (D) and 0.1% of compound 904 (E) is giveninto the mouse anterior tibialis.

Seven days after the injections, expression of the β-galactosidase isobserved by macroscopic visualization of the muscles which receive theformulations in accordance with the present invention. They exhibitregions of fibers expressing β-galactosidase and which become blue afterrevelation by immersing the muscles in a solution containing 2 mM ofMgCl₂, potassium ferricyanide, potassium ferrocyanide, 5 mM of PBS, atpH 7, and in the presence of 1 mg/ml of5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside. The muscles arephotographed 24 h after incubation in this solution at 37° C. Themuscles which received only the naked DNA do not enable β-galactosidaseexpression to be visualized.

FIG. 2 illustrates these results.

EXAMPLE 3 Histological Analysis of the Expression of Green FluorescentProtein (GFP) 7 Days after Injection of 50 μg of Naked DNA (A) or of 50μg of DNA Formulated According to the Invention

The formulations according to the invention that were tested are:

-   -   compound 304 at 5% (w/v) (B);    -   compound 704 at 0.25% (w/v) (C);    -   compound 904 at 0.1% (w/v) (D), and    -   compound 908 at 10⁻³% (w/v).

GFP expression is observed seven days after injection of theformulations, by observing a tissue section mounted between a slide andcover slip and then examined by fluorescence microscopy. FIG. 3 makes itpossible to note that the number of muscle fibers expressing GFP is muchgreater when the DNA is formulated with 304 at 5% (B), 704 at 0.25% (C)and 904 at 0.1% (D). In the particular case of the DNA formulated with908 at 10⁻³% (E), it is noted that the number of transfected fibers isequivalent to that obtained with the naked DNA.

The formulations in accordance with the present invention consequentlyallow a transfection that is at least as efficient as the naked DNA, oreven clearly superior to it. These results confirm the advantage of theclaimed formulations for the expression of a protein of local orsystemic therapeutic interest.

EXAMPLE 4 Comparison of the Efficiency of Transfection into the Muscleof the Formulations in Accordance with the Present Invention, withRespect to Electrotransfer

As regards the electrotransfer conditions, 50 μg of DNA encodingβ-galactosidase were injected into the cranial labial muscle of8-week-old Swiss mice, and the muscles then subjected to the conditionsbelow. As regards the formulations, 50 μg of PCMV-βgal DNA were mixedwith 304, 704 and 904 and then injected into the cranial libial musclesof the mice.

The electrotransfer technique is one of the only non-viral methods whichmakes it possible to improve in vivo transfection in the skeletalmuscle.

The electrotransfer technique considered in the context of thiscomparative test consists in subjecting a muscle to an electric field of200 V/cm with 8 pulses of 20 ms at a frequency of 2 Hz. This test isvalidated through expression of the β-galactosidase in the muscle.

FIG. 4 gives the results obtained. It is noted that the injection ofnaked DNA into the muscle subjected to the electrotransfer makes itpossible to obtain, 7 days after the injection, 10 μg of β-galactosidasesynthesized by the muscle. The formulations of DNA with 304 at 10% (w/v)and of DNA with 704 at 0.25% (w/v) make it possible to obtain thesynthesis, respectively, of 14 and 11 μg of β-galactosidase/muscle.

The formulations of the present invention make it possible to obtain atleast the same transfection efficiency as, or even greater efficiencythan, that of the electrotransferred DNA. What is more, the presentinvention has the clear advantage of not requiring any specificequipment and of being much simpler to carry out since it is sufficientto mix compound (I) with the plasmid DNA. Finally, it is important toemphasize that electrotransfer cannot be applied for gene transfer intocardiac muscle, unlike the formulations in accordance with the presentinvention.

Electrotransfer is, in addition, a painful technique and requiresregional if not general anesthesia.

EXAMPLE 5 Morphology, Visualized by Cryoelectron Microscopy, of ThePoloxamine/DNA Particles as a Function of the Formulation Medium

Poloxamine 904/DNA particles were formulated either with NaCl (150 mM)or with Tyrode's (medium containing 3 mM CaCl₂, 2 mM MgCl₂, 6 mM KCl,140 mM NaCl, 10 mM glucose and 10 mM Hepes, pH 7.4).

For example, measurement of the zeta potential of the poloxamine 904 inthe Tyrode's indicates a value of +9.5 mV, which reflects the cationicuse of the poloxamine when it is combined with the DNA in the presenceof Tyrode's.

The morphology of the particles was then visualized by cryoelectronmicroscopy. FIG. 5 reports the visualization.

When formulated with NaCl (150 mM), the DNA molecules aggregate with oneanother in the presence of poloxamines 904 to form large structures thatare quite “loose”, whereas, with Tyrode's, the DNA molecules form smallspherical particles with the poloxamines 904 (FIGS. 5C, D and E). Thesame observation can be made with poloxamine 304.

Thus, by controlling the pH and the ionic composition of the medium, andin particular the presence of CaCl₂, small spherical particles aregenerated and would probably have an increased capacity for diffusion inthe tissues.

EXAMPLE 6 Influence of the Formulation Medium on the TransfectionEfficiency of the Poloxamine/DNA Formulations in NaCl or Tyrode's

50 μl of formulations containing 15 μg of plasmid DNA encodingluciferase are injected into the anterior tibialis in 8-week-old Swissmice. The muscles are removed 7 days later and ground, and theluciferase activity is assayed.

The table below shows the amount of protein produced by the skeletalmuscle is clearly greater when the formulations are carried out inTyrode's.

Formulation Luciferase Poloxamine medium (ng/muscle) 704.05% (w/v) NaCl94 704.05% (w/v) Tyrode's 625 904.0.01% (w/v)   NaCl 74 904.0.01%(w/v)   Tyrode's 971 904.01% (w/v) NaCl 78 904.01% (w/v) Tyrode's 519

EXAMPLE 7 In Vivo Transfer into the Heart of a Gene Encodingβ-Galactosidase, in the Presence of a Compound of General Formula (I)

Sample Preparation:

Each trial uses 200 μg of plasmid DNA containing the gene encodingβ-galactosidase, and a polymer in 700 μl of Tyrode's.

The polymers PE 6400, for the control sample, and poloxamines 304, 704and 904 were tested at the concentrations (in weight/volume) of 0.5%,2.5%, 0.75% and 0.1%, respectively.

Each sample thus formulated is injected into the pericardial sac ofadult rats weighing approximately 200 g, after having crossed thediaphragm. The hearts are removed 7 days after the injection and areanalyzed by histology in order to reveal the β-galactosidase activity.Only the poloxamine-based formulations make it possible to transfectcardiomyocytes in the left ventricle, and in particular poloxamine 304,which gives the best results. With poloxamine 304, it was observed thatcardiomyocytes expressing β-galactosidase and therefore stained bluewere present to a depth of up to ⅔ in the ventricular mass, which showstheir considerable diffusion capacity.

1. A lyophilized form of a pharmaceutically acceptable injectablecomposition, the pharmaceutically acceptable injectable compositioncomprising: at least one nucleic acid; a Tyrode's medium comprising: 140mM NaCl, 6 mM KCl, 3 mM CaC1₂, 2 mM MgC1₂, 10 mM Hepes, pH 7.4, and 10mM glucose; and a tetrafunctional copolymer of formula (I):

where: x and y represent, independently of one another, an integer ofbetween 1 and 500, and the tetrafunctional copolymer is in a form of amineral salt wherein said tetrafunctional copolymer is in a cationicform; wherein the compound of formula (I) has a molecular weight ofbetween 1,000 and 25,000 g/mol and an EO/PO ratio of 0.8 to 1.2.
 2. Thecomposition of claim 1, wherein the compound of formula (I) is presentin a proportion of 0.005% to 15% by weight/volume.
 3. The composition ofclaim 1, in which the compound of formula (I) is present in a proportionof 0.01% to 10% by weight/volume.
 4. The composition of claim 1, whereinthe compound of formula (I) further comprises at least one intra- orextracellular targeting unit.
 5. The composition of claim 1, wherein thenucleic acid is a deoxyribonucleic acid.
 6. The composition of claim 1,wherein the nucleic acid is a ribo-nucleic acid.
 7. The composition ofclaim 1, wherein the nucleic acid is chemically modified.
 8. Thecomposition of claim 1, wherein the nucleic acid comprises an antisensesequence.
 9. The composition of claim 1, wherein the nucleic acid is aninterfering RNA.
 10. The composition of claim 1, wherein the nucleicacid comprises a therapeutic gene.